Conventional extraction of fucoidan from Irish brown seaweed Fucus vesiculosus followed by ultrasound-assisted depolymerization

Fucoidan has attracted considerable attention from scientists and pharmaceutical companies due to its antioxidant, anticoagulant, anti-inflammatory, anti-tumor, and health-enhancing properties. However, the extraction of fucoidan from seaweeds often involves the use of harsh chemicals, which necessitates the search for alternative solvents. Additionally, the high viscosity and low cell permeability of high molecular weight (Mw) fucoidan can limit its effectiveness in drug action, while lower Mw fractions exhibit increased biological activity and are also utilized as dietary supplements. The study aimed to (1) extract fucoidan from the seaweed Fucus vesiculosus (FV) using an environmentally friendly solvent and compare it with the most commonly used extraction solvent, hydrochloric acid, and (2) assess the impact of ultrasound-assisted depolymerization on reducing the molecular weight of the fucoidan extracts and examine the cytotoxic effect of different molecular weight fractions. The findings indicated that the green depolymerization solvent, in conjunction with a brief ultrasound treatment, effectively reduced the molecular weight. Moreover, a significant decrease in cell viability was observed in selected samples, indicating potential anticancer properties. As a result, ultrasound was determined to be an effective method for depolymerizing crude fucoidan from Fucus Vesiculosus seaweed.


Results and discussion
Conventional extraction was carried out using a GES and 0.1 M HCl to obtain crude fucoidan extracts from Fucus vesiculosus.The extract obtained contains additional biomolecules such as alginic acid, in addition to fucoidan, and is therefore referred to as crude fucoidan.Dried crude fucoidan samples were further treated with 20 kHz US (amplitude 40, 70 and 100%) for 30 min, using different depolymerization solvents (0.1% citric acid, Fenton and distilled water).The depolymerized fractions were subsequently studied for cell viability.

Crude fucoidan
Fucoidan can be extracted from brown seaweeds by various multistage processes using chemical, physical and/ or enzymatic methods, while preserving native properties 19 .The extraction involves diffusion of solvent inside the solid matrix, hydrolysis/solubilisation of target compounds, diffusion of the compounds through the solid matrix and into the bulk solution.
Since the biomolecules are embedded deep in the matrices, it becomes important degrade cell walls, to facilitate the extraction 34 .Hot water and/ or acids (hydrochloric acid, sulphuric acid) or calcium chloride salt are commonly used for extraction of fucoidan 35 .Several factors including temperature, extraction time, pH, liquid-solid ratio and the number of stages involved, influence the extraction of fucoidan 36 .Solvent pH plays an important role in the extraction of fucoidan as it can enhance or hinder the yield of fucoidan obtained.For example, in industrial extraction of fucoidan from Sargassum sp., an increase in pH from 3 to 5 led to a significant increase in fucoidan yield, while a decrease was observed at pH 7. Also pH was found to show a significant interactive effect with temperature and buffer: alga ratio on fucoidan yield (P < 0.05).
It was inferred that low pH can lead to polysaccharide degradation, while high pH can coextract high quantities of alginates which may can increase the solution viscosity and interfere with extraction of fucoidan 37 .From our study crude fucoidan yields of 14.34% and 22.95% were obtained using GES and 0.1 M HCl respectively (Fig. 1).The results demonstrate that HCl resulted in a higher yield of crude fucoidan and is a more efficient extraction solvent compared to GES.Extraction of protein, amino acids and polysaccharides from seaweeds, depends on several factors including type of seaweed, extraction solvent and extraction time.The extraction of polysaccharides from seaweed depends on the solvents that can dissolve the algae cell components and interfere with the hydrogen linkages in the polysaccharide chains 38 .
To obtain fucoidan and alginates from the brown alga Ecklonia radiata 39 performed an acid treatment with HCl solution.This method extracted fucoidan and also enabled the efficient sequential extraction of alginates.The authors reported that the acid disrupts the hydrogen bonds between polysaccharides and facilitates release of fucoidan.The H + of the acid (HCl) can do this more effectively than the GES, which has a lower acidity.Therefore, 0.1 M HCl is a better solvent than GES for extracting polysaccharides from seaweed, as it has a higher extraction yield.However due to safety concerns relating to HCl, edible-grade GES is a viable alternative for fucoidan extraction.
The findings are consistent with a previous study on brown seaweed Sargassum fusiforme 40 , where it was reported that 1 M HCl resulted in more than a two times higher polysaccharide yield compared to using water.Similar findings were reported for Fucus virsoides and Cystoseira barbata 41 , where 0.1 M HCl and 0.1 M H 2 SO 4 resulted in higher polysaccharide yields from Fucus virsoides and Cystoseira barbata compared to using water.

Depolymerization of fucoidan
The Mw of the crude fucoidan samples obtained using the GES and 0.1 M HCl were 399.99 ± 13.28 and 270.43 ± 14.88 kDa respectively.In comparison a Mw of 136.31 ± 5.02 was obtained for the reference commercial fucoidan product analysed.The effect of US treatments (amplitude 40, 70, 100%) and depolymerization solvents (distilled water, 0.1% citric acid and Fenton reagent) on fucoidan samples was analysed.

Effect of ultrasound on Mw reduction of fucoidan samples
All US treatments investigated using selected depolymerization solvents reduced the Mw of fucoidan (Table 1).Differences in Mw of fucoidan samples, depolymerized with US using distilled water, 0.1% citric acid and Fenton reagent are depicted with superscripts a-g, h-o and p-u respectively.At higher US amplitudes increased levels of depolymerization were observed.The maximum reduction in Mw was observed for GES samples subjected to US treatment at 100% amplitude.These results are consistent with a previous study on the depolymerization of raw κ-carrageenan using US, where the authors reported that that high sonication amplitudes and longer treatment time resulted in higher Mw reduction 21 .
Factors that influence the rate of polymer degradation during ultrasonic depolymerization include US intensity, treatment duration and solution concentration.Increasing US intensity generates more cavitation bubbles, which contribute to the degradation process.Ultrasonic degradation is a simple and effective method of polysaccharide depolymerisation, characterised by a high rate of decomposition of large Mw molecules.Cavitation bubbles are formed which can cause intense local heating and high pressure during their collapse.As a result of the bubble collapse, energy is released in the amount sufficient to break the chemical bonds in any polymeric materials.
The rupture of polymer chains as a result of sonolysis occurs in the middle of the molecule, with a greater effect when exposed to low-frequency ultrasound.Increasing US intensity generates more cavitation bubbles, which contribute to the degradation process 19 .
Tecson et al. 21reported that US was efficient in reducing the Mw of all raw κ-carrageenan samples, which can be attributed to the homolytic bond breaking and subsequent reaction with radicals facilitated by the high velocity gradients, temperature (up to 5000 °C), and pressure (about 5 × 10 7 Pa) generated by collapsing cavitation bubbles.When polymers enter the high velocity gradient areas, they are stretched, distorted and stress is generated within the polymer resulting in their bond breakage.In a study involving US and, it was observed that an increase in ultrasonic intensity (424 W/cm 2 ), led to larger reduction in Mw 42 .In another study on US-assisted depolymerization (30 and 80 kHz US frequencies, 30-180 min treatment time) of fucoidan from Sargassum muticum.The authors reported that high US treatment for long time promoted the depolymerization.Also, with 80 kHz, the phenolic content and antioxidant capacity were observed to be increased up to 120 min 43 .
The results are expressed as average ± standard deviation of the mean.The difference in Mw of fucoidan samples, depolymerized with US using distilled water, 0.1% citric acid and Fenton reagent are depicted with superscripts a-g, h-o and p-u respectively.The superscripts representing the impact of US for each solvent, indicate the difference in Mw for US treatments at amplitudes of 40%, 70%, and 100%.For example letters a and b represent the difference between GES crude sample and GES depolymerized sample obtained using distilled water and different ultrasonic amplitudes.P < 0.05.

Effect of solvent on Mw reduction of fucoidan samples
The effect of selected solvent type on the depolymerization of crude fucoidan is shown in Table 2.It was found that depolymerization solvent and US treatment employed influenced the Mw reduction.The Fenton solvent resulted in the highest degree of depolymerization, followed by 0.1% citric acid and distilled water.It has also been reported that US treatment of beta-carotene using 21-25 kHz was limited in reducing the Mw of < 20 kDa due to energy transmission attenuation under a prolonged or high-intensity ultrasonic field 44 .To achieve greater depolymerization, the use of different solvents needs to be studied along with US treatment applied.
US-assisted of pectin has also been studied for pectin.For example, Zhi et al. 45 investigated the depolymerization of pectin using US at 22 kHz and Fenton to produce ultra-low Mw pectin.Their US-Fenton process reduced the 448.26 kDa Mw of pectin to 53.52 kDa in 5 min, and after 35 min the Mw was reduced to 5.5 kDa.In another study 42 , observed that US even in combination with H 2 O 2 failed to degrade the sulfated Table 1.Effect of ultrasound amplitude treatment using distilled water, 0.1% citric acid and Fenton reagent at 30 °C for 30 min on Mw of fucoidan samples.The results are expressed as average ± standard deviation.Statistical differences in Mw of fucoidan samples, depolymerized with US using distilled water, 0.1% citric acid and Fenton reagent are depicted with superscripts a-g, h-o and p-u respectively.prepared rhamnogalacturonon-I enriched low Mw pectic polysaccharide using US (22 kHz, 900 W) and metal free Fenton.They observed that there was a higher reduction in Mw using US/H 2 O 2 /ascorbic compared to H 2 O 2 /ascorbic without US.This shows the synergistic effect of US and the solvent used.However the use of hydrogen peroxide in food industry is limited by regulations in some countries.
In a study reported by Zheng et al. 47 , the authors investigated the impact of US (120 W), hydrogen peroxide concentrations (0.5, 1, 1.2, 1.5%) and 30, 60, 90, 120, 150 and 180 min treatment time on degradation of chitosan.The results indicated that an increase in hydrogen peroxide concentration and US exposure time led to an increase in chitosan degradation.

Cytotoxicity of fucoidan
Higher cytotoxicity activity was observed at higher fucoidan concentrations (Figs. 2, 3, 4).Lower Mw fucoidan samples exhibited higher cytotoxic effects against glioblastoma cells.Depolymerised fucoidan samples which showed a good dose response were analysed using regression analysis to generate IC 50 values (Fig. 5).Li et al. 42 reported that both native and depolymerized fucoidan chondroitin sulfate (fCs-Ib) samples inhibited the viability of A549 lung cancer cells.They reported that US treated low Mw samples showed higher anti-tumour activity than the native (fCs-Ib).Yang et al. 48reported that the biological activities of fucoidans are closely linked to their Mw and sulfate content.Several studies have reported that low-molecular-weight fucoidan (LMWF) is more biologically active than high-molecular-weight fucoidans (HMWF).However, LMWF obtained from acidic hydrolysis leads to reduced bioactivities due to the removal of sulfate groups.Therefore, degrading HMWF into LMWF without removing its functional groups is critical 31 .
Cho et al. 49 studied the effects of sulfation levels in low and high Mw fucoidan compounds on in vitro anticancer activity using human stomach cancer cell line AGS.Fucoidan was partially hydrolysed under mild acid conditions to yield low Mw fucoidan, which was then fractionated by membrane ultrafiltration.High (> 30 kDa) and low (5-30 kDa) Mw fucoidan were observed to be over sulphated.They also found that their over sulphated fucoidan displayed higher (15-30%) anti-cancer activity They reported that the dose dependent antiproliferative activity of the 5-30 kDa Mw fraction against the stomach cancer cell line AGS was two times higher than that of the > 30 kDa fraction.
Cabral et al. 50investigated the influence of Mw fractionation on the antimicrobial and anticancer properties of a fucoidan rich-extract from the macroalgae Fucus vesiculosus.They reported that fucoidan fractions obtained using multiple Mw cut-off (MWCO) have potential to be used as natural and green bacteriostatic and bactericidal ingredients in the food industries.

Discussions
The potential of using green extraction solvent and 0.1 M HCl to extract crude fucoidan from brown seaweed Fucus vesiculosus was demonstrated.Due to safety concerns relating to HCl, edible grade GES is a viable alternative for fucoidan extraction.The method described in this chapter demonstrated a high efficiency in extracting fucoidan from seaweed biomass.This extraction method has a great potential for industrial applications, as it can be scaled-up to produce large quantities of fucoidan.The industry collaborator, Nutramara, uses this method for their commercial production of fucoidan.US treatment at the three amplitudes investigated was effective in depolymerizing high Mw crude fucoidan into low Mw samples for all solvents used.Samples with lower Mw exhibited higher cytotoxic effects compared to high Mw fucoidan samples.This study validated the importance of a suitable solvent along with US for efficient polymer degradation and demonstrated that US can be recommended as an efficient depolymerization method to obtain low Mw fucoidan from Fucus vesiculosus.

Biological material
Seaweed Fucus vesiculosus was harvested from Galway Bay off the coast of Connemara, Co Galway.Collection of plant material was done by Nutramara Ltd, Kerry, Ireland and was in compliance with the national and EU regulations.Seaweeds were harvested and provided as per the licence agreement and Nutramara has the necessary licence/permissions to operate in Ireland.Fucus vesiculosus samples employed in the study are harvested commercially.Details of the samples are available publicly at National Biodiversity Data Centre, Ireland available online https:// maps.biodi versi tyire land.ie/ Speci es/ 187328.The formal identification of the seaweed was undertaken by Dr. Henry Lyons, Scientific Director-Nutramara Ltd., Tralee, County Kerry, Ireland.The samples were dried using an oven dryer at 50-60 °C over 48 h and milled using a hammer mill.Commercially available fucoidan from Fucus vesiculosus was also provided by Nutramara.

Conventional extraction of fucoidan and ultrasound-assisted depolymerization
The seaweed to solvent ratio was kept as 1/10 (w/ v).A green extraction solvent (GES) (pH 3.5) and 0.1 M HCl were used as solvents and conventional extraction at 80 °C, 200 rpm for 2 h was carried out.The seaweed and solvent mixture were kept in a water bath (85 °C) (Clifton range NE1-2.5 unstirred thermostatic bath, UK) and an overhead stirrer (VWR VOS 40 digital) was used to stir the mixture throughout the extraction process (Fig. 6).After 2 h, the mixture was cooled at room temperature and then filtered using a muslin cloth, to separate the residue and the supernatant.The supernatant was mixed with 1% (w/v) calcium chloride and stored at 4 °C.After 24 h, the mixture was centrifuged (3500 rpm, 15 min, 4 °C) using (Sorvall Lynx 6000 centrifuge, Waltham, MA, USA) and the pellets and supernatant were separated.The supernatant was then mixed with 1:3 ethanol (v/v) and stored for 24 h at 4 °C, followed by centrifugation (3500 rpm, 15 min, 4 °C).All the samples (pellets and supernatant) were freeze-dried, at 0.5 mbar, for 2 days using a freeze dryer (Lyovapor™, L-300, Buchi, Flawil, Switzerland).
The crude fucoidan extraction yield was determined by Eq. ( 1) The fucoidan obtained after extraction was subjected to US treatment involving depolymerization solvents (0.1% citric acid, distilled water and Fenton reagent).A Fenton reagent was prepared using 48 mM ascorbic acid and 200 mM hydrogen peroxide solution 40 .Slight modifications were made, along with 40 mL of distilled water, 30 mL of 48 mM ascorbic acid and 30 mL of 200 mM hydrogen peroxide solution was used as the Fenton system.Crude fucoidan (3 g) was mixed with 100 mL depolymerization solvent and US treatments of 20 kHz, amplitudes of 40, 70 and 100% and treatment time of 30 min using US immersion probe, 20 kHz (UIP500hdT, Hielscher www.nature.com/scientificreports/Ultrasonics GmbH, Teltow, Germany) were carried out.The temperature was controlled using a circulation water bath at 30 °C.After the treatments, the sample were freeze-dried and stored at 4 °C under dark conditions prior to further analysis.

Fucoidan molecular weight determination
High performance liquid chromatography coupled with refractive index (HPLC-RI) detector was used to determine the Mw distribution.Fucoidan Mw was quantified using a HPLC system (Agilent 1200 LC system, Agilent Technologies, Santa Clara, California, USA) fitted with a refractive index detector connected to a guard column (OHpak SB-G 6B, 8 × 50 mm) and a Shodex OHpak SB-804 HQ with 6% cross-linked HPLC carbohydrate column of dimensions 8 mm × 300 mm (length × I.D.) (Shodex, Japan) 50 .Samples at a concentration of 2 mg/mL were prepared using the 0.1% NaCl and filtered through 0.45 µm PTFE filters (Econo Filter, Agilent Technologies) and 20 µL were injected into the column using an auto sampler.Separation was achieved using 0.1% NaCl at a constant flow rate of 0.5 mL/min.for 40 min at 40 °C.Mw determination was performed by comparison of the retention times with those of pullulan standard from Sigma (Set Mp ~ 350-700,000, Sigma-Aldrich, St. Louis, MO, USA).The integration of the peaks was performed using the software Agilent Chemstation.A standard curve was developed using different Mw of pullulan.All analysis were performed in duplicate.

Anti-cancer properties
The PrestoBlue cell viability assay was used to assess the cell viability at specific time points post treatment.The PrestoBlue cell viability assay (Thermo Fisher) 51 involves a cell permeable resazurin based solution, which changes colour and becomes fluorescent with the reducing power of living cells, the change is detected by using absorbance or fluorescence measurements.
Cell viability assay 96 well plates were coated with 10 µg/mL laminin for 24 h prior to use.The laminin coating was removed and cells plated at 1 × 10 4 cells per well and left to adhere for 48 h.Fucoidan extracts were reconstituted in fresh media and filter sterilised using a 0.22 µM filter.GCGR-E17 cells were treated with decreasing concentrations from 500 to 0 µg/ mL of fucoidan extracts for 24 h or 5 days.
At the appropriate time point, cell media was removed from each well and replaced with 10% solution of Presto Blue cell viability reagent as per manufactures instructions.Fluorescence was measured with an automated microplate fluorometer (FLUOstar Omega, BMG LabTech) using an excitation wavelength of 544 nm and an emission wavelength of 590 nm.Cell viability was calculated as a percentage of the untreated control.Positive control for the viability assay for 24 h was H 2 0 2 1 mM (24 h) and TMZ 1 mM (5 days).Samples that were untreated were labelled as control.

Statistical analysis
Data was analysed using SPSS version 27 (IBM SPSS Statistics).The treatments were compared with the crude samples and P < 0.05 was used for significance.One-way ANOVA and Tukey test were used to determine the difference.For cytotoxicity, nonlinear regression analysis was carried out using GraphPad Prism V9.

Table 2 .
Effect of depolymerization solvents using ultrasound treatments (40, 70 and 100% amplitude, 30 °C for 30 min) on Mw of fucoidan samples.The results are expressed as average ± standard deviation.Different letters indicate statistical differences in the Mw of fucoidan samples depolymerized using depolymerization solvents at 40%, 70% and 100% US amplitude.The uppercase letters representing the impact of solvent for each US amplitude.For example, difference between Mw of GES crude fucoidan and GES samples obtained with 40% US amplitude and different solvents are labelled as A-B.P < 0.05.